Endogenous tagging of intracellular structures with gene editing​

Figure. Gene-editing versus transient transfection. Z-stacks of live hiPS cells comparing endogenous mEGFP tagging via gene-editing versus overexpression via transient transfection. All movies were imaged with a laser-scanning confocal microscope and images start from the bottom of the cells and end at the top. Movies shows the transient transfections and right panels the gene-edited cell lines for fluorescently tagged α-tubulin (A), desmoplakin (B) and Tom20 (C).​

ObservationsLabeling structures with mEGFP endogenously (via CRISPR/cas9 gene editing) generates human cells that we can image with unprecedented clarity compared to tagged proteins that are generated by transient transfection. The extent of the difference can vary:

Tagged α-tubulin: microtubules are more clearly visible due to decreased background when compared to overexpression

Tagged desmomplakin: desmosomes localize to small puncta near the tops of cells. Transiently transfected cells form more and larger desmosomes or artificial desmosome-like structures.